DE3779578T2 - DEVICE AND METHOD FOR THE MASS TRANSFER OF BIOLOGICAL-PHARMACEUTICAL MEDIA. - Google Patents

Abstract

A mass transfer apparatus and method is based upon use of a mass transfer chamber (12), such as a hollow fiber membrane bioreactor, with means to change the directional flow (16) therethrough for enhancement of the mass transfer operation. The invention has particular utility in cell growth systems, where the periodic reversal of flow direction of a nutrient stream passed through the bioreactor equalizes dispersion of cell growth through the bioreactor and enhances the transport of metabolic waste from the cells being grown.

Description

BACKGROUND OF THE INVENTION Field of the invention

This invention relates to an apparatus and method for mass transfer comprising biological/pharmaceutical media as materials to or from which mass is transferred.

Description of the technology in question

Mass transfer processes are used in numerous applications for the concentration, purification and separation of various biological/pharmaceutical media. Examples include: removal of sodium salts from pharmaceutical compositions to provide low sodium products for human consumption; growth of cell cultures for research; removal of uric acid, creatinine and various undesirable electrolytes from blood via hemodialysis; and the preparation and recovery of viruses for vaccine production, or other intracellular products or secreted biological substances such as immunoglobulins, interleukins, interferons, clotting factors and the like.

In applications of the type described above, it is common practice to use a mass transfer chamber in which a mass transfer surface element is arranged defining passages on its respective sides, wherein the biological/pharmaceutical medium is arranged on one side of the element, and the medium to or from which mass transfer is to be effected is arranged on the opposite side of the element. The respective media are passed in their separate passages over the mass transfer element. contacted with each other, e.g. in countercurrent flow to each other, or wherein one of the materials is retained in its passageway(s) in a batchwise manner while the other material flows past the mass transfer element on the opposite side thereof to effect diffusional and/or osmolytic mass transfer.

A particular problem that has arisen in the operation of such systems is their tendency to experience fouling of the mass transfer element after a period of operation. As an illustration, in systems in which cell media are grown by flowing a nutrient medium on one side of the mass transfer element for diffusional transfer of the nutrient species to a microbial culture on the opposite side of the element, the system experiences after a period of operation preferential areas of cell growth which prevent further mass transfer. In other cases in which proteins are secreted by cell media and subsequently transferred by the mass transfer element to a receiving medium, proteinaceous species deposit on the surfaces of the mass transfer element and create a physical barrier to continued diffusional transfer of the desired products.

These problems are particularly acute in mass transfer surface elements such as hollow fiber bundles of the type wherein a plurality of internal passages accommodate flow of a first medium therethrough, while a second medium is contained in or allowed to flow through the interstitial passages provided between adjacent hollow fibers in the bundle. Such hollow fiber mass transfer elements may have discrete fiber open area dimensions (internal diameter) on the order of about 0.25 to 1 mm, with pores in the mass transfer surface on the order of about 0.2 µ in diameter. Due to the small Dimensions, these mass transfer elements are highly susceptible to clogging during operation and subsequent reduction in the extent and rate of mass transfer.

In addition to hollow fiber membrane modules of the type described above, sponge-like chambers or honeycomb-like modules are variously used. However, the hollow fiber membrane modules are currently the most widely used in cell growth applications, blood dialysis and various other applications.

In the specific area of nutrient mass transfer cell growth systems, a serious problem in the use of the aforementioned mass transfer chambers is the formation of microenvironments within the chambers. Such microenvironments are characterized by poor growth rates, poor longevity of the microbial population, and most notably, areas of active growth within the chamber compared to other areas therein that appear to support little or no growth.

In these chambers, all cell types, both prokaryotic and eukaryotic, when grown as suspension cultures or anchored to a perfusion matrix in the mass transfer chamber, have been observed to grow far more abundantly at one end of the chamber than the other. Neither agitation, such as by using a shaker, nor inverting the chamber to manually manipulate its orientation has solved the problem of the formation of these inappropriate microenvironments.

It has been observed that the preferred growth areas are normally adjacent to the inlet flow from a nutrient source to the mass transfer chamber, and that such active growth at the inlet port effectively causes the following problems: (1) poor utilization of the total potential volume of the chamber for growth;

(2) poor diffusion of nutrient components within these areas of high cell growth; and (3) undesirable accumulation of metabolic wastes in and around the area of high cell growth, which can physically hamper the desired nutrient mass transfer process, particularly if such wastes become deposited on the surfaces of the mass transfer element.

Accordingly, it is an object of the present invention to provide an improved apparatus and method for effecting mass transfer operations involving biological/pharmaceutical media, wherein the degradation of mass transfer accompanying prolonged operation is minimized.

It is another object of the invention to provide a system comprising a mass transfer chamber in which longitudinal degradation of mass transfer efficiency during prolonged operation due to fouling of the mass transfer surface is minimized.

It is another object of the invention to provide an improved apparatus and method for the growth of cells wherein undesirable formation of microenvironments and regions of preferential growth are substantially avoided.

Other objects and advantages of the invention will become more fully apparent from the following disclosure and the appended claims.

SUMMARY OF THE INVENTION

In a broad device aspect, the invention relates to a mass transfer device comprising:

(a) a first pump having an outlet and an inlet channel;

(b) means defining an elongated mass transfer chamber having first and second opposite ends, at least one mass transfer surface element in the chamber having first and second sides each defining first and second passageways, the first passageway(s) on the first side of the element(s) communicating with the first and second ends for flow of a first medium through the first passageway(s) in mass transfer relation with a second medium in the second passageway(s) on the second side of the element(s);

(c) a reservoir having an inlet and outlet opening;

(d) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium is from the first end through the first passage or passages to the second end and a second mode of operation in which the flow of the first medium is from the second end through the first passage or passages to the first end, which comprises switchable flow control means comprising flow openings designated A, B, C and D for connecting:

(1) in a first configuration, opening A is in flow communication with opening B, and opening C is in flow communication with opening D; and

(2) in a second configuration, the opening A is in flow communication with the opening D, and the opening B is in Flow connection with opening C; and

(e) Conduits connecting the pump outlet port to port C, the pump inlet to the reservoir outlet port, the reservoir inlet port to port A, the first end of the chamber to port B, and the second end of the chamber to port D.

The switchable flow control means of the apparatus described above may be any suitable means providing flow communication in the manner aforesaid between the respective conduits in paragraph (e), e.g. a four-way valve, or a series of valves and/or reservoirs etc., and accordingly the term "port" is to be interpreted broadly to include any suitable means of connecting the various conduits so that such switching of flow can be effected. A preferred switchable flow control means is a four-way valve having four ports for connecting the respective conduits.

In another aspect, the apparatus as generally described above comprises at least one mass transfer surface element in the chamber defining passageways on its respective sides, the passageway(s) on one side of the mass transfer element(s) communicating with the first and second inlets for flowing a first medium therethrough in mass exchange relation with a second medium in the passageway(s) on the other side of the mass transfer element(s).

Such a device may further comprise:

(i) an opening on the mass transfer chamber which communicates with the aforementioned second medium passage or passages second medium passages; and

(ii) two valves connected in series at the opening in flow communication with the second medium passage(s), comprising a first valve adjacent to that opening and a second valve connected in series with the first valve, whereby second medium can be introduced into and withdrawn from the mass exchange chamber by flow through the opening and its associated valves connected in series, and the first valve can be closed to retain second medium in the mass exchange chamber while the second valve is open to allow sterilization thereof.

Another aspect of the apparatus described above includes: (1) openings on the mass exchange chamber connected to the second medium passage(s); (2) conduits connecting the openings on the mass exchange chamber to form a second medium flow circuit to which is connected (3) a second pump for circulating the second medium therethrough; and (4) means for intermittently operating the pump according to a predetermined periodic cycle.

In a further aspect, the device described above comprises:

a variable speed stirring device for stirring the contents of the reservoir; and

a control device for the device, comprising:

(I) means for remotely positioning and alternately positioning the switchable flow control device;

(II) a device for remotely varying the speed of the stirring device;

(III) means for remotely varying the pumping rate of the pump connected to the reservoir outlet channel; and

(IV) means for remotely varying the pumping rate of the second pump when actuated by the intermittent actuation means.

A preferred form of such a device comprises as a centralized module a unitary structural housing containing such remote control means (I), (II), (III) and (IV) and the intermittent actuation means.

In one method aspect, the invention relates to a mass transfer method comprising:

(a) Establish a mass transfer system comprising:

(i) a first pump having an inlet and outlet port;

(ii) means defining an elongate mass transfer chamber having first and second ends of the chamber for receiving flow of a first mass transfer medium therethrough in mass transfer relationship with a second mass transfer medium therein, at least one mass transfer surface element being provided in the chamber having first and second sides each defining first and second passageways, the first passageway(s) on the first side of the element(s) being connected to the first and second ends for flow of a first medium through the first passageway or passageways communicate in mass transfer relationship with a second medium in the second passageway or passageways on the second side of the element or elements;

(iii) a first mass transfer medium reservoir having an inlet and outlet opening;

(iv) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium is from the first end through the first passage or passages to the second end and a second mode of operation in which the flow of the first medium is from the second end through the first passage or passages to the first end, comprising switchable flow control means comprising flow openings designated A, B, C and D for connecting:

(1) in a first configuration, opening A in fluid communication with opening B, and opening C in fluid communication with opening D;

(2) in a second configuration, the opening A in fluid communication with the opening D, and the opening B in fluid communication with the opening C;

(v) Pipes connecting the pump outlet port to port C, the pump inlet port to Connect the reservoir outlet port, the reservoir inlet port to port A, the first inlet of the chamber to port B, and the second inlet of the chamber to port D;

(b) operating the first pump to establish a flow of the first medium through the mass transfer chamber in a selected direction; and

(c) periodically repositioning the switchable flow control device so that the direction of flow of the first medium through the mass transfer chamber is sequentially changed according to a predetermined pattern.

By way of illustration, the second medium may be selected from the group consisting of cell cultures, blood and pharmaceutical compositions.

Other method aspects of the invention include those corresponding to the operation of the various device aspects of the invention described above. A particularly preferred method aspect of the invention relates to a cell growth method comprising:

(a) Establish a sterile growth system comprising:

(i) a pump having a discharge port and an inlet port ;

(ii) means defining an elongate cell growth chamber having first and second ends at opposite ends of the chamber, at least one mass transfer surface element in the chamber having first and second sides defining respective first and second passageways, the first passageway the first passages on the first side of the element or elements having the first and second ends communicating for flow of a first medium through the first passage or passages in mass transfer relationship with a second medium in the second passage or passages on the second side of the element or elements;

(iii) a culture medium reservoir having an inlet and outlet opening;

(iv) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium is from the first end through the first passage or passages to the second end and a second mode of operation in which the flow of the first medium is from the second end through the first passage or passages to the first end, comprising switchable flow control means comprising flow openings designated A, B, C and D for connecting:

(1) in a first configuration, opening A in fluid communication with opening B, and opening C in fluid communication with opening D;

(2) in a second configuration, the opening A in fluid communication with the opening D, and the opening B in fluid communication with the opening C;

(v) conduits connecting the pump outlet port to the port C, the pump inlet port to the reservoir outlet port, the reservoir inlet port to the port A, the first inlet of the chamber to the port B and the second inlet of the chamber to the port D;

(b) introducing culture medium into the system;

(c) seeding the chamber with a selected cell type;

(d) operating the pump to establish a flow of the medium through the growth chamber in a selected direction;

(e) incubating the system for a selected period of time and changing the medium during that period of time as and as required for the selected medium; and

(f) periodically repositioning the switchable flow control device so that the direction of flow through the growth chamber is sequentially changed according to a predetermined pattern to distribute cell growth substantially uniformly throughout the chamber and to transport metabolic waste away from cells grown in the chamber.

Other aspects and features of the invention will become more fully apparent from the description set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of a cell culture system containing a four-way valve according to the invention.

Figure 2 is a schematic drawing of the cell culture system of Figure 1 with a remote control arrangement for the four-way valve.

Figure 3 is a schematic drawing of the four-way valve in a first position.

Figure 4 is a schematic drawing of the four-way valve in a second position.

Figure 5 is a schematic drawing of a mass transfer system which may be usefully used in dialysis-type extractions of blood components or for desalting pharmaceutical compositions.

Figure 6 is a schematic drawing of a cell growth system of a general type as shown in Figure 2, but modified by the further addition of a sterile barrier means for introducing or buffering nutrient solution and for oxygenating the nutrient solution on its return to the reservoir, and in which a uniform remote control means is used.

Figure 7 is a plan view of a unitary remote control device of the type that may be conveniently used in systems of the type shown in Figure 6.

Figure 8 is a rear elevational view of the device of Figure 7.

Figure 9 is a schematic representation of elements of the Remote control system and the system components controlled by it.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

Having repeatedly observed that cell growth in cell and culture media mass transfer systems is directly related to the inlet of fresh culture media, it was hypothesized that changing the direction of flow of the nutrient source to the chamber would result in greater uniformity of growth within the chamber. Additional factors observed were that the increased agitation of such a flow scheme would increase the recirculation of metabolic waste as well as increase the homogeneity with respect to pH and gas exchange in the cell medium. A successful device and method is based on the use of a reversible flow control device, preferably a four-way valve, which allows the direction of fluid flow to be reversed by activation of one valve alone.

Referring now to the drawings, Figure 1 shows an illustrative nutrient delivery cell growth system 10 utilizing a conventional type hollow fiber membrane 12 as the bioreactor. The bioreactor comprised a bundle of hollow fibers, e.g., from 1 to about 3 inches in diameter, presenting on the order of 60% open area in cross-section. The inner diameter of individual fibers in the bundle may be, e.g., from about 0.25 to about 1 mm, with pores for diffusional and/or osmolytic transfer of nutrient species in the wall of the tubular fibers of about 0.2 µm diameter.

Illustrative hollow fiber membrane mass transfer elements suitable for the practice of the invention are available from A/G Technologies, Needam, MA and Cordis Corporation, Miami, FL.

The hollow fiber membrane 12 is connected at one end, designated E-1, by conduit 14 to an opening designated D, which is one of four such openings of a four-way valve 16, the others being designated A, B and C. The conduit may be a flexible, resilient silicone tube, or it may be formed of a more rigid material, such as 316 stainless steel tubing. The opposite end of the hollow fiber membrane 12, designated E-2, is connected by conduit 18 to the opening B of the four-way valve 16. A four-way LL valve 16 generally useful in the general practice of the invention is manufactured by the Quality Controls Company, Tilton, New Hampshire.

The bioreactor system shown in Figure 1 also includes a variable speed pump 20, which may include, for example, the peristaltic pump 20 illustrated in Figure 1, as is commercially available from the Cole-Palmer Company, Chicago, Illinois, or a variable speed gear pump, such as the MICROPUMP™ gear pump, which is commercially available from the MICROPUMP Corporation, Concord, CA.

The pump 20 is connected on its discharge side by line 22 to port C of the four-way valve 16. The inlet side of the pump 20 is connected by line 24 to the outlet port of a reservoir 30 which contains the nutrient medium for the bioreactor system. Such a reservoir may be of a type commercially available from the Bellco Company, Vineland, New Jersey. The inlet end of the reservoir 30 is connected by another line 32 to port A of the four-way valve 16.

The reservoir 30 is arranged on a magnetic stirrer device 35, which stirs the contents of the reservoir The magnetic stirrer may suitably be of a variable speed type to provide a varying level of agitation in the reservoir 30 depending on the density and suspension properties of the nutrient medium contained therein.

The mass transfer chamber comprising the hollow fiber membrane 12 also features a first opening 38 proximate the inlet E-1 and a second opening 40 adjacent the inlet E-2. These openings may be provided with suitable closure means or they may be connected to a suitable flow circuit, as described in more detail hereinafter, for circulating the cell medium contained in the hollow fiber membrane 12.

Accordingly, in this embodiment, the cell medium is disposed in a set of passages in the membrane 12 which are defined by the interstitial spaces between adjacent hollow fibers in the bundle. Such bundles are typically potted at their ends in urethane or epoxy resins so that they can be suitably headed to accommodate flow through the internal passages of the hollow fibers without leakage into the interstitial passages. Nutrient is thus flowed longitudinally through the internal passages of the hollow fibers and the nutrient species is transferred through the fiber walls to the cell culture contained in the interstitial passages of the bundle.

The four-way valve 16 has two positions. In what is referred to as a first position, the port C is connected to the port D and the port B is connected to the port A. In this mode of operation, the nutrient soup in the line 24 is caused to flow from the reservoir 30 by means of the pump 20 into the line 22 from which it enters the port C, discharges through the port D into the line 18 and enters the hollow fiber membrane bioreactor at its E- 2. At the same time, the contacted nutrient soup exits through the opposite end, designated inlet E-2, of the mass transfer chamber and passes through ports B and A of the four-way valve 16 to be returned through line 32 to the inlet of reservoir 30 from which the nutrient medium is circulated in the manner previously described.

When the four-way valve 16 is in what is referred to as its second position, port C is connected to port B and, accordingly, the valve discharges the nutrient soup into line 14 from which it enters the mass transfer chamber containing the hollow fiber membrane at the inlet designated E-1, passes through the internal passageways of the hollow fibers and exits at the opposite end of the mass transfer chamber designated E-2 for return in line 18 through ports D and A of the four-way valve 16 and line 32 to the reservoir 30.

Switching of the four-way valve can be accomplished either manually or by an automatic actuator, as illustrated schematically in Figure 2, in which the valve 16 is controlled by a suitable automatic controller 54 operatively connected to the valve assembly by control signal wires 56. Continued operation of the bioreactor system with repositioning of the valve 16 either manually or by remote control, e.g., from a pneumatic or electric actuator, thus results in a balanced delivery of nutrients to the mass transfer chamber alternately from its respective ends. Periodically switching the flow in this manner according to a predetermined cyclic schedule also increases the transport of metabolic waste away from the cells. Furthermore, the increased agitation associated with switching the nutrient flows results in a more homogeneous environment with respect to other metabolic parameters.

Except for the four-way cyclic valve switching mechanism previously described, the system of Figure 2 is identical in all respects to that of Figure 1 except for the provision of two valves in series at each of the ports 38 and 40. At the first port 38, the valve assembly 42 includes a first valve 46 adjacent the port and a second valve 44 connected in series with the first valve, as shown. A similar construction is used at the second port 40 where the dual valve assembly 48 includes a first valve 52 and a second, outer valve 50.

The dual valve arrangement described in the preceding paragraph is highly preferred in practice because it allows for cell inoculation of the interstitial passages in the hollow fiber bundle as well as withdrawal of the product cell culture from the interstitial passages in a manner that makes it possible to maintain complete sterility of the device. Specifically, in the previous practice of using ported hollow fiber cell growth chambers, only one valve has been used on each port. In such a single valve arrangement, the introduction of cell medium into or withdrawal of cell medium from the mass transfer chamber results in loss of sterility of the internal surfaces of the valve, thereby increasing the potential for contamination of the mass transfer chamber unless the device is then completely shut down and the valves removed and autoclaved. Although some attempts have been made in the past to sterilize single-valved ports by directing superheated steam against the valve body structure, such a sterilization approach does not reach the internal surfaces of the valve, which must be closed to retain the contents in the mass transfer chamber.

In contrast, the double valve arrangement shown allows easy sterilization of the valve arrangement in a Manner which maintains the sterility of the associated device by the simple expedient of opening the outermost valve (42 or 48) while keeping the corresponding inner valve (46 or 52) closed, and superheated steam or other sterilant is passed into the opened outer valve to effect complete sterilization thereof. In this manner, a sterile barrier is maintained at the mass transfer chamber even during continued operation and without the need to shut down the apparatus for treating the valve elements in an autoclave. Although simple in character, this dual valve construction is a significant advance in the art due to the criticality of sterility in cell growth as well as in numerous other mass transfer operations in which bioreactors of the type shown in Figures 1 and 2 may be employed.

Figures 3 and 4 are simplified schematic representations of the internal flow passages of an illustrative four-way valve of the general type described in connection with Figures 1 and 2. Figure 3 shows the four-way valve in a first position in which the inner member 17 of the valve 16 is oriented so that the valve ports B and A are in fluid communication with each other and the ports C and D are in fluid communication with each other. By rotating the valve member 17 90°, the configuration shown in Figure 4, referred to as the second position, is achieved. In this position, the valve ports B and C are in fluid communication with each other while a fluid communication is established between the ports A and D in a corresponding manner. Referring again to the systems schematically illustrated in Figures 1 and 2, it can be seen that changing the valve position between the respective configurations shown in Figures 3 and 4 causes a cyclically alternating switching of the nutrient medium flow to the respective ends of the mass transfer chamber containing the hollow fiber membrane 12.

Although the mass transfer chamber has been described with specific reference to a hollow fiber bundle as the mass transfer surface element, it is within the scope of the invention to use other mass transfer elements, such as planar membranes, through which mass transfer can be effected, it being further understood that the number of specific passageways within the mass transfer chamber can be varied widely depending on the particular mass transfer media and application being used.

Figure 5 is a schematic drawing of a mass transfer system that can be used for mass transfer operations other than the cell growth processes illustratively described in connection with the embodiments of Figures 1 and 2. For example, the device of Figure 5 can have utility for mass transfer operations such as: desalting pharmaceutical compositions; desalting, extracting or fractionating blood, plasma or serum materials; and recovering secreted or intracellularly produced biological materials such as interferons, interleukins, immunoglobins, growth factors, coagulation factors, etc.

In Figure 5, all system elements corresponding to those shown in Figures 1 and 2 are numbered accordingly by adding 100 to the corresponding reference numbers of Figures 1 to 2.

In this mass transfer system, the mass transfer chamber contains a hollow fiber bundle 112 which provides a first set of internal passages in the hollow fibers forming the same through which a first medium supplied from reservoir 130 is allowed to flow. Simultaneously, a second mass transfer medium stored in a second reservoir 180 is withdrawn in line 182 to which peristaltic pump 184 and discharged into line 186 from which it enters the mass transfer chamber at port 190 and flows longitudinally through the interstitial passages in the hollow fiber bundle. After such longitudinal flow in which the second medium is contacted with the first mass transfer medium which is simultaneously flowed through the internal passages of the hollow fiber bundle, the contacted second medium is discharged from the mass transfer chamber at port 192 and allowed to flow in line 188 back to the second reservoir 180. A sterile barrier at the mass transfer chamber may be provided by dual valve assemblies 142 and 148 as shown on the third and fourth ports 138 and 140 of the mass transfer chamber, respectively.

Figure 6 shows a modification of the cell growth system shown in Figure 2, wherein corresponding system elements have been numbered accordingly with reference to Figure 2 by adding 200 to the reference numbers of the same or corresponding elements of Figure 2.

As in the embodiment of Figure 2, the mass transfer chamber includes a hollow fiber bundle 212 having internal flow passages within the hollow fibers communicating with respective inlets E-1 and E-2 of the chamber, which in turn are connected by lines 214 and 218 to ports B and D of the valve assembly 216, respectively. Port C of the valve assembly is connected to line 222 and port A to line 232a to provide reversing flow through the mass transfer chamber upon switching of the valve assembly in the manner described above. The valve assembly includes an actuator which is operated by control signals transmitted in signal wires 256 from the unified remote control 254, as described in more detail hereinafter.

As in the system of Figure 2, the system shown in Figure 6 has System includes first and second ports 238 and 240 on the mass transfer chamber to which dual valve assemblies 242 and 248 are connected, respectively. The mass transfer chamber further includes a third port 260 and a fourth port 261 connected to an external flow circuit comprising lines 262 and 264, respectively, whereby the cell culture or other second medium can be withdrawn from the interstitial passageways of the hollow fiber bundle 212 and circulated through the external flow circuit by means of the pump 263 connected thereto.

The remote control unit 254 is connected in control relationship to the peristaltic pump 263 by means of control signal transmission wires 265 so that the pump 263 is periodically activated and operated for a predetermined time to circulate the cell mass outside the mass transfer chamber. This causes redistribution and agitation of the cell mass and significantly increases the mass transfer efficiency of the system, resulting in a more uniform distribution of cell growth in the hollow fiber bioreactor. For example, in some cell systems it has been found advantageous to operate the pump 263 on a 6 minute cycle time schedule, which includes active pumping for periods on the order of about one minute in duration and motionless intervals between active pumping during which the pump 263 is deactivated of approximately five minutes.

The remote control unit 254 is also operatively connected to the peristaltic pump 220 via control signal transmission wires 266 and to the magnetic stirrer 235 via control signal transmission wires 267.

Such an arrangement allows the peristaltic pumps 220 and 263, the four-way valve assembly 216 and the magnetic stirrer 235 to all be controlled from a single remote location, thereby simplifying the design and operation of the system simplified relative to the use of multiple remote control devices.

As in the system of Figure 2, nutrient soup is held in the reservoir 230 from which it is circulated in line 224 by the pump 220 into line 222 for flow into the inlet end E-1 or E-2 of the mass transfer chamber, depending on the position of the four-way valve assembly 216.

Depending on this valve position, the effluent nutrient soup from the mass transfer chamber is discharged into either line 214 or 218 and moved to reservoir 230 via return flow line 232, which includes line sections 232a and 232b.

In this return flow line 232, which comprises the line sections 232a and 232b, a second hollow fiber mass transfer chamber 2 is arranged, which has an inlet end 4 and an outlet end 3, which is constructed analogously to the previously described mass transfer chamber comprising the hollow fiber membrane 212. The second mass transfer chamber is provided with a first opening 5, which is connected to a double valve arrangement 7, and a second opening 6, to which a double valve arrangement 8 is connected.

At its outlet end 3, the second mass transfer chamber is connected to the inlet 61 of a third mass transfer chamber 60, which comprises a hollow fiber mass transfer element, by a suitable connecting device 9 of a conventional type known in the art. The outlet end 62 of the third mass transfer chamber is in turn connected to the aforementioned conduit section 232b.

The third mass transfer chamber 60 has a first opening 65 to which an oxygen gas supply device 63 is connected via the connecting line 64. By such an arrangement, oxygen is supplied from the supply device to the interstitial passages of the hollow fiber bundle of the third mass transfer chamber 60. This chamber has a second opening 66 provided with a flow control valve 67 connected to a discharge line 68 for controllable discharge of contacted gas therefrom.

In operation, the nutrient soup that has been contacted with the cell culture in the mass transfer chamber comprising the hollow fiber membrane 212, before being returned to the reservoir 230, flows in line 232a to the second mass transfer chamber 2, the double-valved openings of which provide a sterile barrier against external contamination of the system. By such means, the reservoir 230 can be maintained in a fully closed condition during operation of the system, and additional nutrient materials and/or additives to the nutrient soup can be supplied to the system through the openings 5 and/or 6 for flow into the interstitial passages of the second mass transfer chamber and diffusional and/or osmolytic transfer into the nutrient soup that is returned to the reservoir in the internal passages of the hollow fiber bundle. Accordingly, the returned nutrient soup can be buffered, cleaned, etc. by means of the second mass transfer chamber without breaching the intended sterile barrier. After introducing or removing medium through the openings 5 or 6, the opening can be sterilized by opening the outer valve of the dual valve assembly while the inner valve is held in the closed position and introducing a sterilizing agent onto the inner and outer surfaces of the outer valve in the manner previously described.

For the growth of aerobic cell cultures in the first mass transfer chamber comprising the hollow fiber bundle 212, it is important to maintain an appropriate dissolved oxygen content of the nutrient soup supplied by the reservoir 230. Oxygen enrichment of the nutrient medium is effected in the system of Figure 6 by the third mass transfer chamber 60. Oxygen gas from source 63 is introduced into line 64 and enters the third mass transfer chamber at port 65 from which it enters the interstitial passages in the hollow fiber bundle and is transferred to the internal passages of the hollow fiber bundle containing the flowing nutrient medium. Oxygen-depleted gas from the mass transfer contacting process is discharged from the third chamber through port 66 and flows into discharge line 68 as controlled by flow control valve 67. The flow control valve 67 may be feedback controlled by suitable coupling to an oxygen sensing device, such as a probe for dissolved oxygen in the nutrient soup in the reservoir 230 or an oxygen sensing gas analyzer used to sense the oxygen content of the gas exhausted from the line 68, whereby the flow control valve 67 can be adjusted in response to the oxygen concentration sensing so that the oxygen content of the nutrient soup in the reservoir 230 is maintained at a predetermined level.

Figures 7 and 8 show the structural features of the unified remote control unit 254 schematically illustrated in Figure 6. In this remote control unit, the electronic control circuit is contained in a housing which includes a top portion 302, a bottom portion 310, side walls 304 and 306, and a rear wall 308.

On the upper part 302 there is arranged a control panel 316, which includes an on-off switch 318 for the magnetic stirrer control 320, which can be manually adjusted to set a predetermined rotational speed for the magnetic stirrer 235 (see Figure 6).

The on-off switch 330 turns on the valve timing control 332, which can be manually set so that it varies the switching frequency of the four-way valve assembly 216 of Figure 6. In practice, it has been found advantageous to design the valve timing control to provide switching intervals over a range of 1 minute increments from one to five minutes so that the flow of nutrients through the cell growth mass transfer chamber can be reversed periodically at and from such predetermined time intervals according to a selected schedule of operation for the given system.

The on-off switch 322 energizes the pump control 324, which is manually adjustable to set a predetermined rotational speed for the pump 220 in Figure 6.

Similarly, the on-off switch 326 energizes the pump control 328, which is manually adjustable to provide a predetermined rotational speed for the pump 263.

Figure 8 shows the details of the rear panel 308 of the remote control unit. The remote control unit is supported, as shown, on casters 312 and 314, which may be made of a resilient material such as neoprene rubber, to dampen the vibrations of the system and prevent damage to the electronic circuitry contained within the unit.

Electrical power is supplied to the electronic circuitry in the unit by means of a power cable connection socket 336 adjacent to which is a fuse holder 338 containing a suitably rated fuse for protecting the electronic circuitry. A main switch 334 provides electrical energization of the circuitry in the unit.

On the right side of the rear panel 308, as shown in Figure 8, there are suitable electrical sockets for connecting the Remote control unit is provided with the control signal transmission wires for the controlled elements of the system. Accordingly, socket 340 receives a plug connected to signal control wires 365, socket 342 receives a plug connected to signal control wires 266, socket 344 receives a plug connected to signal control wires 267, and socket 346 receives a plug connected to signal control wires 256, reference being made to the control wires as identified in Figure 6.

Figure 9 is a schematic electrical block diagram of the remote control system and associated controlled elements.

Power to the remote control unit is supplied in power lines 400 and 401 (as connected to the remote control unit through an associated connector in socket 336 as shown in Figure 8).

Power from the main power lines is transmitted to the variable speed controller 414, from which electrical control signals are transmitted in wires 266a and 266b to the pump 220.

Branch power lines 408 and 409 convey power to timing device 410 which is connected by control signal wires 411 and 412 to variable speed control 413 which in turn is connected by signal wires 265a and 265b in signal control relationship to pump 263. Branch lines 415 and 416 convey electrical power from lines 408 and 409 respectively directly to variable speed control 413. In such a manner, variable speed control is energized during the period which the timing device determines for active operation of pump 263, consistent with the above-described use of pump 263 to periodically circulate nutrient soup in the external flow circuit which lines 262 and 264 (see Figure 6). During the intervening idle periods when the external flow circuit is not being used, the timing device 410 receives power from the branch lines 408 and 409 but does not operate to actuate the variable speed control.

Branch power lines 405 and 406 supply power to the speed controller 407, which is connected to the magnetic stirrer 235 through signal control transmission wires 267a and 267b, whereby a predetermined rotational speed of the magnetic stirrer can be effected.

Finally, branch power lines 402 and 403 supply power to the timing device 404 which is connected by means of the signal transmission wires 256a and 256b in signal control relationship to the four-way valve assembly comprising the valve actuator 216a and the valve 216b.

The specific construction and arrangement of the electronic circuitry for the variable speed controls and timing devices may be conventional in character and may vary widely in specific features, it only being necessary that such circuitry be suitably unified when used in a unitary remote control, as illustratively shown in Figures 7 and 8.

The features and advantages of the invention will be more fully demonstrated by reference to the following examples, in which all parts and percentages are by weight, unless expressly stated otherwise.

EXAMPLE I

A hollow fiber bioreactor system 10 was constructed as previously described in Figure 1. This system was tested under Chemically sterilized using hypochlorite solution and rinsed with sterile deionized water. The system was then filled with culture medium, RPMI 1640 with 5% fetal calf serum, and incubated for 48 hours at 37ºC in a 5% carbon dioxide, no humidity environment as a control for sterility and system equilibrium.

An inoculation of the EL-4-IL-2 cell type was prepared from stock cultures and inoculated into the extracapillary space of the hollow fiber bioreactor. The system was again incubated for 20 days with media changed as required by previous practice for this cell type. The four-way LL valve 16 was switched every half hour during the working day and once every night, usually around midnight, taking care to use a different positioning on consecutive nights.

The particular cell culture used had previously been grown in membrane bioreactors and had been observed to grow closer to the inlet end. In this experiment, the four-way LL valve 16 was used to evenly distribute cell growth throughout the growth chamber 12. The cell type used is generally referred to as a suspension culture, which allows for easy visualization of cell growth by simple liquid turbidity observation.

EXAMPLE II

A simple hollow fiber bioreactor system 10 was constructed, as previously described in Figure 1. This system was then filled with culture medium, MEM with Earle's salts, non-essential amino acids and 10% fetal calf serum, and incubated for 48 hours at 37ºC in a 5% carbon dioxide, no humidity environment as a control for sterility and system equilibrium.

A seed of BALB-3T3 cell type was prepared from stock cultures and seeded into the extracapillary space of the hollow fiber bioreactor. The system was then incubated for an additional 30 days, changing media as required by previous practice for this cell type. The four-way LL valve 16 was switched every half hour during the working day and once each night, usually around midnight, taking care to use alternating positioning on consecutive nights.

The culture used in this example had previously been grown in membrane bioreactors where it had been observed that a predominance of cell nodules, indicative of cell proliferation, was formed primarily at the nutrient inlet. When the cells were cultured in the bioreactor system 10 using the four-way LL valve 16, nodules were visible throughout the hollow fiber module. In addition, the hollow fiber module was slit open and individual fibers were examined microscopically with and without the aid of stain, hematoxylin and eosin, and uniform cell proliferation was observed at both ends of the fibers.

EXAMPLE III

A hollow fiber bioreactor system 110 was constructed as shown in Figure 6. The system was autoclave sterilized except for the hollow fiber oxygenator 60 which was aseptically inserted into the nutrient return line as shown. The system was then filled with a culture medium, modified Frey's medium supplemented with 8% porcine family serum, pH 7.0, through the sterile barrier tangential flow device 2 and incubated for 48 hours at 37°C in ambient air as a control for sterility and system equilibrium. A 20 milliliter 0.1% cell suspension of Mycoplasma galasepticum was inoculated into the extracapillary space (interstitial passageways) of the hollow fiber bioreactor 112. The system was then incubated for 35 days with media changed as required based on the cell densities extracted from the extracapillary space. The four-way LL valve 216 was automatically cycled every minute by a timing device 34 manufactured by Eutectic Electronic, Raleigh, NC.

Mycoplasma species are routinely grown in suspension cultures at densities of 0.4% cells or less and have an affinity for fibrous membranes and plastics. Therefore, the hollow fiber bioreactor 212 used in this experiment, supplied by Applied Membrane Technologies, Minnetonka, Minnesota, had two major modifications: 1) a polyolefin coating was placed on the 0.1 micron base membrane to counteract the tendency of the mycoplasma to adhere to the membrane fibers, 2) the extracapillary space had four exit ports. Two of the diagonally opposite ports were connected by tubing through a Cole-Parmer peristaltic pump 263 as shown in Figure 6. The pump was used to ensure complete uniformity of the culture by preventing adhesion of the organisms to the fibers and to provide additional mixing to the extracapillary space nutrients.

The extracapillary space pump 263 was evaluated in two different modes of operation: (1) a continuous slow recirculation of the nutrient medium; and (2) an intermittent mode of operation of 1 minute on and 5 minutes off. The second mode was hypothesized to be less resolving of overall protein stability. The recirculation rate for pump 263 was set to one system volume per minute.

Mycoplasma cells were the desired product of these experiments. Special emphasis was placed on being able to obtain a uniformly dispersed cell suspension with the highest possible cell densities.

Several preliminary experiments were carried out using this organism with remarkable results. In the first experiment without a four-way LL valve 216 or extracapillary pump 263, a cell suspension of roughly 1 to 2% was observed growing and adhering to the fibers at the inlet port of the bioreactor 12. A second experiment using the four-way LL valve 216 but not the extracapillary pump 263, cell densities of 4.0% were achieved. The organisms were evenly distributed along the length of the bioreactor 212, but continued to be in close proximity to the fibers. This adhesion of the organisms to the fibers made obtaining a uniform suspension difficult. In the final experiment, using the four-way valve and external flow circuit components as shown in Figure 6, yields of uniformly dispersed 10% cell suspensions were routinely obtained at 12-hour intervals.

In the above experiments, a hollow fiber oxygenator 60, commercially available from CD Medical Corporation, Miami Florida, was used along with a Biostat M Fermentor (B. Braun, West Germany) as the culture medium reservoir 230. The oxygenator 60 was used to facilitate oxygenation of the culture medium rather than squirting through the medium in the reservoir 230. No notable change in growth was expected or observed due to the oxygenator 60, which was added for convenience and evaluation purposes only. The B. Braun Fermentor was used to overcome a lack of available warm room space for temperature control and to provide automatic rather than manual pH monitoring and control. as a test of overall system integrity when operated in a manufacturing setting. The increased cell growth observed was not attributable to the automatic pH control, in that the pH monitor and pH control were turned off for several days to directly test this hypothesis.

A sterile barrier tangential flow device 2, available commercially from Microgon Inc., Laguna Hills, CA, was used for all media changes in the second and subsequent experiments. The increased safety provided by this sterile barrier tangential flow device was highly beneficial. The final mycoplasma experiment was carried out using the aforementioned fermentor without the benefit of a sterile hood or other confining device. There were more than seventy media changes during this experiment without loss of sterility.

An additional measure was taken for sterility in all of the aforementioned experiments on Mycoplasma. This involved the use of two serially connected two-way valves (Becton Dickenson) on all hollow fiber membrane cartridge ports. This dual valve arrangement allowed for resterilization of the outermost valves with alcohol on plastic valves or flame on stainless steel valves while the inner valve remained closed, thus ensuring a sterile barrier. The dual valve arrangement was essential for collecting cells from the extracapillary space of bioreactor 212. The dual valves 242 and 248 made it possible to maintain a sterile environment in the extracapillary space, despite over 100 samples being drawn during the last experiment alone.

From the above it can be seen that the device and method of the invention provide a relatively simple bioreactor system which allows the uniform distribution of the Cell growth is dramatically improved throughout the growth chamber, the transport of metabolic waste away from the cells is increased, and a more homogeneous environment for other metabolic parameters is created throughout. Accordingly, the primary objectives of the invention have been achieved. Other incidental advantages include the ability to use relatively simple tubing which is easy to assemble and maintain. Another practical advantage is the minimal number of connections required, which reduces the potential occurrence of sterility breaches. It is also recognized that the flow of culture medium through the growth chamber could be controlled in a more complicated manner by providing a plurality of valves, however, control of a single valve is inherently more desirable and accurate than controlling multiple valves. Further, valve failure of the four-way valve in the system of the invention would not itself cause cell death.

Although specific embodiments and features of the invention have been described in detail, it is recognized that other variations, modifications and embodiments thereof are possible, and accordingly, all such variations, modifications and embodiments are considered to be within the spirit and scope of the invention.

Claims (29)

1. A mass transfer device comprising: (a) a first pump having an outlet and an inlet port; (b) means defining an elongated mass transfer chamber having first and second opposite ends, at least one mass transfer surface element in the chamber having first and second sides each defining first and second passages, the first passage(s) on the first side of said element(s) communicating with the first and second ends for flow of a first medium through the first passage(s) in mass transfer relation with a second medium in the second passage(s) on the second side of said element(s); (c) a reservoir having an inlet and outlet opening ; (d) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium is from the first end through the first passage or passages to the second end, and a second mode of operation in which the flow of the first medium from the second end through the first passage or passages to the first end, which comprises a switchable flow control device comprising flow openings designated A, B, C and D, for connecting: (1) in a first configuration, opening A is in flow communication with opening B, and opening C is in flow communication with opening D; and (2) in a second configuration, the opening A is in flow communication with the opening D, and the opening B is in flow communication with the opening C; and (e) Conduits connecting the pump outlet port to the port C, the pump inlet to the reservoir outlet port, the reservoir inlet port to the port A, the first end of the chamber to the port B, and the second end of the chamber to the port D. 2. Device according to claim 1, wherein the switchable flow control device is manually positionable. 3. Device according to claim 1 or 2, comprising a device for positioning the switchable flow control device. 4. Apparatus according to claim 3, wherein the positioning means alternately positions the switchable flow control means in the first and second configurations, according to a predetermined periodic cycle. 5. Apparatus according to any one of claims 1 to 4, wherein the first pump comprises a peristaltic pump. 6. Apparatus according to any one of claims 1 to 4, wherein the first pump comprises a gear pump. 7. Apparatus according to any one of claims 1 to 6, wherein the mass transfer surface element comprises a hollow fiber membrane. 8. Apparatus according to any one of claims 1 to 7, comprising means for flowing the second medium through the second passageway(s) on the second side of the element(s). 9. Apparatus according to claim 8, wherein the means for flowing the second medium through the second passage or passages on the second side of the element or elements comprises: (i) first and second openings on the mass exchange chamber communicating with the second medium passageway or passageways; (ii) a second medium reservoir; (iii) conduits connecting the second medium reservoir to the first and second openings on the mass transfer chamber to form a second medium flow circuit; and (iv) a second pump connected to the second medium flow circuit for circulating the second medium therethrough. 10. Device according to claim 1, comprising: (i) an opening on the mass transfer chamber communicating with the second medium passage or passages; and (ii) two valves connected in series at the opening in flow communication with the second medium passageway or passageways, comprising a first valve adjacent to said opening and a second valve connected in series with the first valve; whereby second medium can be introduced into and withdrawn from the mass exchange chamber by flow through said opening and its associated valves connected in series, and the first valve can be closed to retain second medium in the mass exchange chamber while the second valve is open to allow sterilization thereof. 11. Apparatus according to claim 10, comprising a second said opening on the mass exchange chamber communicating with the second medium passage(s) and having said two series-connected valves connected thereto. 12. Device according to claim 10 or 11, comprising: (1) a third and fourth opening on the mass exchange chamber communicating with the second medium passageway or the second medium passageways; (2) conduits connecting the third and fourth openings on the mass exchange chamber to form a second medium flow circuit; (3) a second pump connected to the second medium flow circuit for circulating the second medium therethrough; and (4) means for intermittently operating the pump according to a predetermined periodic cycle. 13. Apparatus according to claim 1, comprising means defining a second elongate mass transfer chamber having first and second opposite ends connected to the conduit connecting the reservoir inlet to the flow port A, thereby positioning the second mass transfer chamber in series with and intermediate the reservoir inlet and the flow port A, the second mass transfer chamber having at least one valved opening for introducing externally supplied media thereto and withdrawing internally contained media therefrom. 14. Apparatus according to claim 13, comprising means defining a third elongate mass transfer chamber having first and second opposite ends connected to the conduit carrying the Reservoir inlet connected to the opening A so that the third mass transfer chamber is arranged in series relationship intermediate between the second mass transfer chamber and the reservoir inlet, and gas supply means connected to the third mass transfer chamber for gas contacting medium which is caused to flow therethrough to the reservoir. 15. Device according to claim 13 or 14, comprising: a variable speed stirring device for stirring the contents of the reservoir; and a control device for the device, comprising: (I) means for alternately positioning the switchable flow control device; (II) means for varying the speed of said stirring means; (III) means for varying the pumping rate of the first pump; and (IV) means for varying the pumping rate of the second pump when actuated by the intermittent actuating means. 16. Apparatus according to claim 15, comprising as a centralized control module a unitary structural housing containing the control means (I), (II), (III) and (IV) and the intermittent actuation means. 17. Apparatus according to any one of claims 1 to 16, wherein the switchable flow control device is a four-way valve. 18. Mass transfer process comprising: (a) Establish a mass transfer system comprising: (i) a first pump having an inlet and outlet port; (ii) means defining an elongate mass transfer chamber having first and second ends of the chamber for receiving flow of a first mass transfer medium therethrough in mass transfer relationship with a second mass transfer medium therein, at least one mass transfer surface element being provided in the chamber having first and second sides each defining first and second passages, the first passage(s) on the first side of the element(s) communicating with the first and second ends for flow of a first medium through the first passage(s) in mass transfer relationship with a second medium in the second passage(s) on the second side of the element(s); (iii) a first mass transfer medium reservoir having an inlet and outlet opening; (iv) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium occurs from the first end through the first passage or passages to the second end and a second mode of operation in which the flow of the first medium occurs from the second end through the first passage or passages to the first end, comprising a switchable flow control device which comprises flow openings designated A, B, C and D for connecting: (1) in a first configuration, the opening A is in flow communication with the opening B, and the opening C is in flow communication with the opening D; and (2) in a second configuration, the opening A is in flow communication with the opening D, and the opening B is in flow communication with the opening C; (v) conduits connecting the pump outlet port to the port C, the pump inlet port to the reservoir outlet port, the reservoir inlet port to the port A, the first inlet of the chamber to the port B and the second inlet of the chamber to the port D; (b) operating the first pump to cause a flow of the first medium through the mass transfer chamber in a chosen direction; and (c) periodically repositioning the switchable flow control device so that the direction of flow of the first medium through said mass transfer chamber is sequentially changed according to a predetermined pattern. 19. The method of claim 18, comprising flowing the second medium through the second passageway(s) on the second side of the element(s). 20. A method according to claim 18 or 19, wherein the second medium is periodically circulated from the mass transfer chamber through an external flow circuit communicating with the second passageway(s) on the second side of the element(s). 21. A method according to any one of claims 18 to 20, wherein the second medium is selected from cell cultures, blood or pharmaceutical compositions. 22. A method according to any one of claims 18 to 21, wherein the mass transfer chamber is provided with an opening which communicates with the second passage(s) on the second side of the element(s), two valves connected in series are connected to said opening, comprising a first valve adjacent to said opening and a second valve adjacent to the first valve, comprising sterilising the second valve in an open position while the first valve is closed. 23. A method according to any one of claims 18 to 22, comprising providing a supply reservoir of the second medium and pumping the second medium from the second medium supply reservoir through the chamber and back to the second medium supply reservoir. 24. The method of claim 21, wherein the second medium is a cell culture and the first medium is a nutrient medium therefor. 25. The method of claim 24, wherein the first medium flowing through the conduit connecting the reservoir inlet to the opening A is an oxygen-enriched upstream flow of the reservoir. 26. The method of claim 18, comprising: (d) periodically pumping the second medium from the mass transfer chamber through an external flow circuit; (e) stirring the first medium in the reservoir; and (f) Remote control of: Pumping rate in steps (b) and (d); Periodicity of the pumping step (d); Valve positioning in step (c); and Stirring rate in step (e). 27. The method of claim 26, wherein the remote control steps are performed at a single remote location. 28. A method according to any one of claims 18 to 27, wherein the switchable flow control device is a four-way valve. 29. A cell growth method comprising: (a) Establish a sterile growth system comprising: (i) a pump having an outlet and an inlet port; (ii) means defining an elongated cell growth chamber having first and second ends at opposite ends of the chamber, at least one mass transfer surface element in the chamber having first and second sides each defining first and second passageways, the first passageway(s) on the first side of the element(s) communicating with the first and second ends for flowing a first medium through the first passageway(s) in mass transfer relation with a second medium in the second passageway(s) on the second side of the element(s); (iii) a culture medium reservoir having an inlet and outlet opening; (iv) means for alternately switching the direction of flow of the first medium through the first passage or passages between a first mode of operation in which the flow of the first medium occurs from the first end through the first passage or passages to the second end and a second mode of operation in which the flow of the first medium occurs from the second end through the first passage or passages to the first end, comprising a switchable flow control device comprising flow openings designated A, B, C and D for connecting: (1) in a first configuration, the opening A is in flow communication with the opening B, and the opening C is in flow communication with the opening D; and (2) in a second configuration, the opening A in flow communication with the opening D and the opening B in flow communication with the opening C; (v) conduits connecting the pump outlet port to the port C, the pump inlet port to the reservoir outlet port, the reservoir inlet port to the port A, the first inlet of the chamber to the port B and the second inlet of the chamber to the port D; (b) introducing culture medium into the system; (c) seeding the chamber with a selected cell type; (d) operating the pump to establish a flow of the medium through the growth chamber in a selected direction; (e) incubating the system for a selected period of time and changing the medium during that period of time as and when required for the selected medium; and (f) periodically repositioning the switchable flow control device so that the direction of flow through the growth chamber is sequentially changed according to a predetermined pattern to distribute cell growth substantially uniformly throughout the chamber and to transport metabolic waste away from cells grown in the chamber.